As epithelial cells progress to carcinomas, increased autocrine TGF-? signaling acquires a prominent role in cancer progression, by inducing an epithelial plasticity response that can lead to epithelial-mesenchymal transition (EMT). EMT results in cell de-adhesion and increased cell motility and invasion, a prerequisite of cancer cell dissemination, and is increasingly seen as an integral property of carcinoma stem cells. As TGF-? signaling drives EMT, and TGF-? responsiveness contributes to cancer progression, we have been studying the regulation of TGF-? signaling, as it pertains to epithelial plasticity. The well-studied Smad signaling pathway regulates gene expression in response to TGF-?, but the TGF-?- induced epithelial plasticity response cannot be explained merely by changes in gene regulation. Accordingly, TGF-?-induced non-Smad signaling has received increasing appreciation. Supported by this grant, we have been studying the TGF-?-induced activation of the Erk MAPK and PI3K-Akt-mTOR pathways, and have started addressing their roles in EMT. The specific roles of TGF-?-induced activation of either pathway in the epithelial plasticity response remain to be further defined. We also found that cells regulate their responsiveness to TGF-?, by regulating the TGF-? receptor levels at the cell surface from intracellular stores. Increased glucose levels and insulin activate this upregulation of cell surfae TGF-? receptors, which appears to be mediated by Akt activation and the Rab GTPase activating protein AS160, a direct target of Akt phosphorylation. We hypothesize that increased Akt activation, as commonly seen in carcinomas, or resulting from increased glucose or insulin stimulation, enhances the cell's TGF-? responsiveness, and the sensitivity and susceptibility of cancer cells to EMT, and thus may promote cancer progression by enhancing TGF-? responsiveness. We now seek to continue our research program aimed at characterizing the roles of non-Smad signaling mechanisms in the control of the cell surface TGF-? receptor levels, and resulting TGF-? responsiveness, and in TGF-?-induced EMT. We organized our current and future research in three Aims: (1) To study the effects of glucose or insulin on TGF-? signaling, epithelial-mesenchymal transition, cancer stem cell generation and EMT-dependent cancer progression; (2) To define the molecular mechanisms regulating the cell surface presentation of the TGF-? receptors in response to Akt activation; (3) To define the roles of TGF-?-induced Erk MAPK and PI3K-Akt pathway activation in epithelial-mesenchymal transition, and cancer stem cell generation. Our studies should provide novel mechanistic insights into the regulation of TGF-? responsiveness and the roles of TGF-?-induced non-Smad signaling in the cellular TGF-? response, in particular in EMT and cancer stem cell generation. These insights may link hyperglycemia or insulin treatment with cancer progression, through increased TGF-? responsiveness, and reveal a new role for the increased Akt signaling that is commonly seen in carcinomas, thus contributing to cancer progression by enhancing TGF-? responsiveness.